Co-Q is a quinone derivative which plays an important role in the terminal electron transport system of organisms. The structure of Co-Q is 2,3-dimethoxy-5-methyl-1,4-benzoquinone having an isoprenoid side chain at the 6 position, and there are various homologs including Co-Q.sub.6 -Co-Q.sub.10 depending on the number of the isoprene unit in the side chain. Co-Q occurs in various kinds of organisms and those in yeasts are known to be Co-Q.sub.6 -CoQ.sub.10. Their physiological activity and pharmacological effects recently have been elucidated.
Co-Q hitherto has been produced by extraction from animals and plants and partly by chemical synthesis, but the cost is so high that industrial production on a large scale has been difficult. Therefore, attempts to produce Co-Q by fermentation recently have been attempted. However, the Co-Q content of cells produced by the usual method of cultivating microorganisms is rather low. There is a method in which a yeast of the genus Candida is cultivated in a medium containing n-alkane with the addition of a precursor of Co-Q such as p-hydroxybenzoic acid to increase the Co-Q content of cells (Japanese Pat. No. 673,128). However, in this method, the amounts of Co-Q produced per culture broth are small and the carbon source is limited to n-alkane.
In order to carry out the fermentative production of Co-Q, the present inventors extensively examined species of yeast, the composition of media and cultural conditions, and have developed a cultivation method by which yeast cells containing large amounts of Co-Q are produced in high concentrations.
The production of Co-Q with use of microorganisms must be considered with respect to both the Co-Q content of cells and the cell productivity from the point of view of the production cost. So far, in the production of Co-Q using yeast, the cultivation method which attached importance to an increase in the Co-Q content sacrificed the productivity of cells whereas, on the other hand, the employment of the usual method for efficiently producing cells resulted in a decrease in the Co-Q content of cells.
The present inventors considered that the maintenance of highly oxidative conditions during cultivation would exert a favorable influence on the formation of Co-Q in view of the important role of Co-Q in oxidative reactions in organisms. In order to maintain the oxidative state of the environment in which cells grow, the concentration of dissolved oxygen in a culture medium must be raised. An increase in the oxygen supply or a decrease in the oxygen consumption will lead to an increase in dissolved oxygen in a culture medium. The present inventors investigated the relationship between the concentration of dissolved oxygen in a culture medium (hereinafter referred to as D.O.) and both the Co-Q content of cells and the cell productivity with various yeasts, and have found that yeast cells produced under conditions of high D.O. contain large amounts of Co-Q.
Many methods for the efficient production of yeast cells are known, for example, one in which aeration and agitation are increased in order to raise the rate of oxygen supply; one in which the partial pressure of oxygen in aerating gases is increased; one in which cultivation is carried out under elevated pressures; and one in which the cultivation apparatus is improved. However, an increase in oxygen supply to the culture broth in the usual cultivation processes for the production of cell mass leads to an increase in cell productivity (hereinafter referred to as .mu.x:g. cells produced/l/hr) and an increase in the oxygen consumption proportional to the increase in .mu.x and, as a result, owing to decreased D.O., it is difficult to keep the oxidative environment suitable for the formation of Co-Q. The Co-Q content of yeast cells grown under conditions of maintaining D.O. values at not less than 2 ppm by supplying sufficient amounts of oxygen for the growth of cells in disregard of the production cost is sometimes higher than that of yeast cells cultivated at D.O. of not more than 1 ppm. But an increase in D.O. does not necessarily raise the Co-Q content of yeast cells, depending on kinds of yeasts, cultural conditions (temperature and pH) and the composition of media.
Cultivating yeast while regulating D.O. at not less than 2 ppm and changing cultural conditions and the composition of media, the present inventors investigated the relation of the Co-Q content of cells to the cell concentration (hereinafter referred to as X), the specific growth rate (hereinafter referred to as .mu.) and cell productivity (.mu.x) at each cultivation time, and have found that there is a clear correlation between the Co-Q content of cells and .mu.. The Co-Q content of cells tends to increase when a yeast which exhibits a maximum specific growth rate of not less than 0.15 hr.sup.-1 under the optimum cultural condition is cultivated while the average specific growth rate (.mu. from the beginning to the end of cultivation) is controlled by means of cultural conditions and the composition of media. Especially when the yeast is cultivated at the average specific growth rate of not more than 0.1 hr.sup.-1, the Co-Q content of cells is markedly increased.
The optimum cultural conditions somewhat vary according to the kinds of yeasts, but generally are presented as follows:
Cultivating Apparatus: jar fermentor; PA1 Medium: glucose 20 g/l, peptone 10 g/l, and yeast extract 5 g/l; PA1 Temperature: 25.degree.-35.degree. C. PA1 Agitation: 700 rpm; PA1 Aeration: 2 vvm. PA1 t=cultivation time, hr.
The present inventors have further examined in detail the relation of the Co-Q content to D.O., .mu.,.mu.X, and methods for regulating .mu., and have succeeded in developing a cultivation method favorable for the Co-Q production. The present inventors have found that yeast cells containing Co-Q in large quantities are efficiently produced by maintaining the average specific growth rate of not more than 0.1 hr.sup.-1 and D.O. of not less than 2 ppm, and completed the present invention based on this finding.